The present invention is in the field of lipase proteins that are related to the lysosomal acid lipase subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides novel peptides and proteins that effect protein phosphorylation and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.
Lipases
The lipases comprise a family of enzymes with the capacity to catalyze hydrolysis of compounds including phospholipids, mono-, di-, and triglycerides, and acyl-coa thioesters. Lipases play important roles in lipid digestion and metabolism. Different lipases are distinguished by their substrate specificity, tissue distribution and subcellular localization.
Lipases have an important role in digestion. Triglycerides make up the predominant type of lipid in the human diet. Prior to absorption in the small intestine, triglycerides are broken down to monoglycerides and free fatty acids to allow solubilization and emulsification before micelle formation in conjunction with bile acids and phospholipids secreted by the liver. Secreted lipases that act within the lumen include lingual, gastric and pancreatic lipases, each having the ability to act under appropriate pH conditions. Modulating the activity of these enzymes has the potential to alter the processing and absorption of dietary fats. This may be important in the treatment of obesity or malabsorption syndromes such as those that occur in the presence of pancreatic insufficiency.
Lipases have an important role in lipid transport and lipoprotein metabolism. Subsequent to absorption across the intestinal mucosa, fatty acids are transported in complexes with cholesterol and protein molecules termed apoliporoteins. These complexes include particles known as chylomicrons, very low density lipoproteins (xe2x80x9cVLDLsxe2x80x9d), low density lipoproteins (xe2x80x9cLDLsxe2x80x9d) and high density lipoproteins (xe2x80x9cHDLsxe2x80x9d) depending upon their particular forms. Lipoprotein lipase and hepatic lipase are bound to act at the endothelial surfaces of extrahepatic and hepatic tissues, respectively. Deficiencies of these enzymes are associated with pathological levels of circulating lipoprotein particles. Lipoprotein lipase functions as a homodimer and has the dual functions of triglyceride hydrolase and ligand/bridging factor for receptor-mediated lipoprotein uptake. Severe mutations that cause LPL deficiency result in type I hyperlipoproteinemia, while less extreme mutations in LPL are linked to many disorders of lipoprotein metabolism.
Lipases have an important role in lipolysis. Free fatty acids derived from adipose tissue triglycerides are the most important fuel in mammals, providing more than half the caloric needs during fasting. The enzyme hormone-sensitive lipase plays a vital role in the mobilization of free fatty acids from adipose tissue by controlling the rate of lipolysis of stored triglycerides. Hormone sensitive lipase is activated by catecholamines through cyclic AMP-mediated phosphorylation of serine-563. Dephosphorylation is induced by insulin. While mice with homozygous-null mutations of their hormone-sensitive lipase genes induced by homologous recombination have been shown to enlarged adipocytes in their brown adipose tissue and to a lesser extent their white adipose tissue, they are not obese. White adipose tissue from homozygous null mice retain 40% of their wild type triacylglycerol lipase activity suggesting that one or more other, as yet uncharacterized, enzymes also mediate the hydrolysis of triglycerides stored in adipocytes. Hormone-sensitive lipase does not show sequence homology to the other characterized mammalian lipase proteins.
As identified above and in the cited references, lipase proteins are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of the lipase family of proteins. The present invention advances the state of the art by providing previously unidentified human proteins that have homology to known members of the lipase family of proteins.
Lysosomal acid lipase (LAL) hydrolyzes cholesteryl esters and triglycerides that are delivered to the lysosomes by low density lipoprotein receptor-mediated endocytosis.
Molecular cloning of a full-length cDNA for human lysosomal acid lipase/cholesteryl ester hydrolase (EC 3.1.1.13) reveals that it is structurally related to previously described enteric acid lipases, but lacks significant homology with any characterized neutral lipases.
Its amino acid sequence, as deduced from the 2.6-kilobase cDNA nucleotide sequence, is 58 and 57% identical to those of human gastric lipase and rat lingual lipase, respectively, both of which are involved in the preduodenal breakdown of ingested triglycerides. Notable differences in the primary structure of the lysosomal lipase that may account for discrete catalytic and transport properties include the presence of 3 new cysteine residues, in addition to the 3 that are conserved in this lipase gene family, and of two additional potential N-linked glycosylation sites.
The human LAL cDNA and chromosomal gene have been characterized, and the locus maps to human chromosome 10q23.2-23.3(3, 4). The gene has 10 exons spread over 36 kilobase pairs. Several hLAL mutations have been detected in cDNAs derived from mRNAs of CESD and WD patient cells. In CESD, a splice donor site G to A transition leads to aberrant splicing of exon 8 and a 72-base pair (24-amino acid) deletion. An AG deletion leads to frameshift at amino acid 302 (AG302) and a truncated lipase with a 34-amino acid C-terminal deletion. Two proline to leucine substitutions (L179P and L336P) have been detected in different CESD patients. In WD, a T insertion at nucleotide 635 results in a frameshift (fs177) and premature translation termination at amino acid 189.
Deficient activity of lysosomal acid lipase (LAL) is associated with two autosomal recessive traits which expressed as two major phenotypes: Wolman disease and cholesterol ester storage disease (CESD). Wolman disease occurs in infancy and is nearly always fatal before the age of 1 year. Hepatosplenomegaly, steatorrhea, abdominal distension, adrenal calcification, and failure to thrive are observed in the first weeks of life. On the other hand, CESD has a more benign clinical course. CESD may not be detected until adulthood, and moderate hyperbetalipoproteinemia, hypertriglyceridemia, and hepatomegaly may be the only clinical signs. In addition, low plasma HDL cholesterol (HDL-C) and premature atherosclerosis occur in most cases with CESD. The enzymatic defect has been demonstrated in several types of cells and tissues, including liver, spleen, lymph nodes, aorta, peripheral blood leukocytes, and cultured skin fibroblasts. Higher residual activity of LAL in intact fibroblasts was found from patients with CESD than in those with Wolman disease, providing a biochemical explanation for the less severe phenotype associated with CESD.
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Lipase proteins, particularly members of the lysosomal acid lipase subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of this subfamily of lipase proteins. The present invention advances the state of the art by providing a previously unidentified human lipase proteins that have homology to members of the lysosomal acid lipase subfamily.
The present invention is based in part on the identification of amino acid sequences of human lipase peptides and proteins that are related to the lysosomal acid lipase subfamily, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate lipase activity in cells and tissues that express the lipase. Experimental data as provided in FIG. 1 indicates expression in the colon tumor, colon, kidney renal cell adenocarcinoma, placenta, teratocarcinoma, skin melanotic melanom, B cell from burkitt""s lymphoma and mixed pool tissues of brain, heart, kidney, lung, spleen, testis, leukocyte.